Breathing Manganese and Iron: Solid-State Respiration

Breathing Manganese and Iron: Solid-state Respiration KENNETHH. NEALSON Center for Great Lakes Studies University of Wisconsin at Milwaukee Milwaukee, Wisconsin 53204

BRENDALITTLE Naval Research Laboratory Stennis Space Center Stennis, Mississippi 39529 I. Introduction 11. Respiration: Organismal and Environmental A. Aerobic Respiration B. Anaerobic Respiration C. Environmental Respiration 111. Metal-Reducing Bacteria in Captivity IV. Reduction of Metals by Iron and Manganese Reducers A. General Features B. Biochemistry of Metal Reduction C. Regulation of Metal Reduction D. Products of Metal Reduction V. Electron Transport In Metal Reducers A. Cytochromes B. Quinones VI. Metal-Reducing Bacteria in Natural Environments A. Corrosion B. Release of Adsorbed Pollutants C. Clay Reduction D. Bioremediation VII. Summary References

I. Introduction

This review concerns a relatively new concept in microbial metabolism: the use of solid metal substrates by bacteria that grow anaerobically, using iron and manganese oxides as oxygen substitutes for respi2 13 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 45 Copyright D 1997 by Academic Press All rights of repruduction in any form reserved. OOfi5-2164/97 $25.00

214

KENNETH H. NEALSON AND BRENDA LITTLE

ration. Recognition of the organisms and their metabolism is new, but the knowledge that such respiration occurs in the environment has been taught for many years in environmental science and oceanography. Measurements of carbon turnover and porewater chemistry have demonstrated that iron and manganese are potent oxidants of organic matter in sediments (Froelich et a]., 1979) and anoxic water columns (Nealson et al., 1991). Data from these environments [Fig. 1) indicative of some kind of anaerobic respiration led several groups to attempt isolation of metal-reducing bacteria (MRB). In this review the concept of respiration as an organismal and environmental process is discussed, with attention to how geochemical (environmental) data suggest the presence of MRB. There follows a discussion of the current state of knowledge concerning MRB, the problems they pose, and the potential uses they may represent.

II. Respiration: Organismal and Environmental A. AEROBIC RESPIRATION

Aerobic respiration is the process whereby electron transport occurs from a fuel (reduced compound) to oxygen, and in the process protons are pumped across a membrane to create a chemiosmotic gradient, or proton motive force (PMF). The PMF is then used to generate biologically useful energy in the form of adenosine triphosphate (ATP), or to run other cellular processes directly. For eukaryotes the oxidant is molecular oxygen, and respiration is usually assessed by direct measurement of oxygen consumption. Most readers should be comfortable with this concept of respiration. In general, the higher forms of life on earth use organic carbon as an energy source and molecular oxygen as the oxidant to burn the organic fuel. With a few notable exceptions, eukaryotes cannot survive for long periods in the absence of molecular oxygen; their metabolism is geared to aerobic respiration, and in its absence the organisms suffocate. There are some exceptions: a few fungi exist anaerobically via fermentation, and some anaerobic protists (ciliates and flagellates) have anaerobic bacteria as symbionts and are capable of exploiting anaerobic niches via these partners. The concept of breathing is, of course, intimately connected to this view of respiration, as oxygen is a gas, and many eukaryotes have special organs specifically for the exchange of gas and the associated breathing process.

LAKE MICHIGAN (FreshwaterSediment)

BLACK SEA (Marine Basin) % max

Yomax

I 3-4

50 I

100

I

1

02

sod'

.. MnTT II

5

k

10

Fett

-t E I

I

k W

W 0

a

15

20 NOZ-

10.3pM; NH4+ = 120 pM;SO4' = 300 pM; CHI = 300 pM

NO2-= 10.1 pM; NH~+=30pM;SO~==25rnM;HiS=100pM

FJG.1. Gradients of electron donors and/or acceptors in anoxic stratified environments. This diagram shaws porewater nutrients from freshwater (Lake Michigan) sediment on the left, and water column measurements of the same nutrients horn a stratified marine environment [Black Sea). The numbers are as a percentage of the maximum value seen for such environments, with the 100% values shown at the bottom.

KENNETH H. NEALSON AND BRENDA LITTLE

216

TABLE I CLASSICAL ELECTRONACCEPTORS FOR B ACXKRIAL RESPIRA.I.ION

Chemical statc Gases

Solutions

Product

Oxidant

Prokary-

Eukaryotic

+

otic

co,

Oxygen

Water (H20) Methane (CH,)

+ +

Nitrate (NO,) Nitrite (NO,)

Nitrogen (N,) Nitrogen (N,)

+ +

Sulfate (SO:-) Polysulfide (So) sulfite SO,^-)

Sulfide [H,S) Sulfide (H,S) Sulfide (H2S)

+ + +

TrimethylamineN-oxide (TMAO)

Trimethyl nniine

b .

Dimethylsulfoxide (DMSO]

Dimethylsulfide

+

Fumarate

Succinate

+

Glycine

Acetate

+

B. ANAEROBIC RESPIRATION

In marked contrast to the eukaryotes, prokaryotes are notorious for survival under anoxic conditions, with a major mode of metabolism being that of anaerobic respiration. In this metabolic mode, any of a variety of inorganic and organic compounds may be used as “oxygen substitutes” for the oxidation of organic carbon: some are dissolved gases, while others are dissolved salts of solids (Table I). The concept of breathing is somewhat altered in these situations, as the prokaryotes have no special organs or organelles for gas exchange. However, the concept of respiration with dissolved electron acceptors other than oxygen has been known for many years and has offered no particular conceptual problems to biologists or biochemists. Thus, both the substrates shown in Table I and the organisms that use these substrates for anaerobic respiration are well known (see Zehnder, 1988).

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

217

C. ENVIRONMENTAL RESPIRATION

In many physically stabilized environments, where mixing and convection are minimal phenomena, gradients of chemicals form due to the activity of microorganisms, and quite often the gradients are the direct result of respiratory activities. Concentration profiles show oxygen depletion for both a Lake Michigan sediment and the Black Sea stratified water column (Fig. 1).In the freshwater sediment, anoxia occurs about 2 cm below the aerobic sediment-water interface. From that point downward, breakdown of organic carbon is accomplished either via fermentation or anaerobic respiration, and environmental gradients reflect these processes. For example, after oxygen depletion, a zone of nitrate and nitrite depletion develops, followed by zones of manganese and iron reduction, and finally a zone of methane production (COz reduction). In the Black Sea water column, oxygen depletion occurs at approximately 50 m, followed by zones of nitrate, nitrite, manganese, and iron reduction. In marine systems, however, sulfate reduction, rather than methanogenesis, predominates, leading to the appearance of sulfide in the deep zones. In lake systems, where sulfate concentration is low, sulfate is rapidly consumed, and the remaining organic carbon is disposed of via COz reduction to methane. Thus, the high concentration of sulfate in seawater (Table 11) leads to sulfur-dominated marine systems as compared to their methanedominated freshwater counterparts. Figure 1 demonstrates a fact known by marine chemists for many years and discussed in detail by Froelich et al. (1979); namely, that respiration observed in the environment involves some components not normally considered by bacteriologists-that is, the role of metals as oxidants of organic carbon. In both systems shown in Fig. 1,there is an appearance of MnZ+and Fez+, and for most stratified environments throughout the world, similar profiles can be constructed (Davison, 1993; Nealson and Saffarini, 1994). The profiles are a measure of the respiratory activity of the environment-oxidizing equivalents that are being reduced at the expense of the oxidation of organic carbon in nature. It was observations like these that led to the interest by microbiologists in organisms that could respire solid metal substrates. Arguing from general principles, one might expect that manganese or iron could act as an electron acceptor for the oxidation of organic matter for two reasons. First, both have redox potentials that should allow them to be utilized in preference to many other electron acceptors; second, both are often found in abundance in sedimentary environments (Table 11).The redox potential of manganese is near that of nitrate,

218

KENNETH H. NEALSON AND BRENDA LITTLE TABLE I1 COMI'AKISON OF

Electron acceptor

ELECTRON ACCEPTORS AVAILABLE IN THE ENVIRONMENT

p") [W]"

Natural ahundanre (pM)

Electron acc/cm;

Free energy (kJ/M gliicosc)

0 2

+13.75

300'

4.3

-3190

NO; Mn4+ Fe

+12.65

10-20'

n0.85

-3030

+8.9

<1 to >1000d

e2.6

-3090h

-0.80


4

-1410'

250 (FW)

112

-380

Variahld

1

-350

'+

so;-

-3.5

CO,

-4.13

>inoo"

28000 (Mar)P

"This is the electron activity for unit activities of oxidant and reductant at neutral pH, as calculated by Zchndcr and Stumm (1966). "'l'he solubility of oxygcn is tcmpcraturc sensitive. but this is a rcasonable number for natural walers 'Nitrate is usually quite low, with these numbers representing high values, except in polluted areas. dBoth Mn a i d Fe form iusoluble oxidized tornis, so thal in water coluinns they tend to be very low (less than 1 pM), hut in sediments they can reach 111714values or Iiigher. "Sulfate ~:~inr:entration varies widely in trashwater e n v i n i n ~ ~ ~ el,ul ~ i l susually does not exceed 200-300 pM, while in marine environments i t lypic:ally is at 28 mM. /COz varies widely, depending 011 alkalinity, nrgmic carlmn inpul, eti: ,?Thisnumber is based on the oxitlalion ol"'standard" organic carbon, as discussed by Froelich et a / . (1979).

l q . 1 11s ' iitiinIx:r can wry, ~lnp~:iiding on the Mn oxide (Nnnlson and Saffarini, 1994). Wiis numher can vary, dopcnding on thc Fc oxide (Nealson and Saffarini, 1994).

while that of iron is lower, hut still significantly above that of sulfate. Thus, in studies of environmental respiration, King (1990) demonstrated that addition of iron or manganese oxides inhibited the respiration of sulfate. While indirect effects could have led to the same results, direct interaction of these oxidants with bacteria could not he dismissed. Using different approaches, several workers have attempted to assess the importance of iron and/or manganese in environmental respiration, and in some cases these metals are major redox components. For example, Aller (1990) and Aller et al. (1991) showed that oxidized manganese was the major electron acceptor in sediments fiom the Amazon shelf, often accounting for 70% or more of the organic carbon that was respired. Similarly, Canfield ef al. (1993) have shown that Mn recycles many times in offshore sediments and can account for

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

219

virtually all of the organic carbon oxidized in some sediments. Aguilar and Nealson (1994) measured the flux of reduced Mn2+from sediments of Oneida Lake (NY) and calculated the rate of oxidation of carbon oxidation, using a stoichiometry of approximately 2.5:l (Mn2+produced per COz produced, as shown in Table 11). The authors calculated that a few percentage points of the annual primary productivity of this eutrophic lake was remineralized via Mn4+oxidation or environmental respiration of this metal. In summary, the study of “environmental respiration” led to the discrepancy between the known “classical” oxidants for respiration (Table 1) with those observed to be active in the environment (Fig. 1). While Mn4+and Fe3+reduction were major environmental processes, there were no known bacteria that could live by respiration of iron or manganese. Even now, the concept of dissimilatory metal reduction is not common in microbiology textbooks. The reason for this discrepancy can be traced to the beliefs of bacterial physiologists that oxides and oxyhydroxides of iron and manganese are solids, and that solids are unavailable for direct reduction by bacteria. Thus, any interactions that occurred were assumed to be via indirect reduction by soluble reductants: the reduction of the metal oxide by an indirect, nonenzymatic process. There is little doubt that such indirect reactions can and do occur (Stone, 1987a,b),as shown for the reduction of Mn(1V) by sulfate reducers (Burdige and Nealson, 1986) or iron reducers (Myers and Nealson, 1988b), but there is also no doubt that dissimilatory reduction is a process common to several groups of bacteria and of possible importance in a variety of environments and processes. It is these organisms, the dissimilatory metal-reducing bacteria (MRB or DMRB), and their metabolism that will be discussed in this review-organisms that have found a niche on solid metal oxide surfaces, where they rapidly reduce the metal oxides and grow at the expense of organic carbon oxidation. Ill. Metal-Reducing Bacteria in Captivity

In 1988, three publications reported dissimilatory reduction of iron and/or manganese by pure cultures of bacteria. The growth of the facultative anaerobe Shewanella putrefaciens (a.k.a. Alteromonas p u trefaciens; MacDonell and Colwell, 1985) on iron (DiChristina et a]., 1988) and manganese (Myers and Nealson, 1988a) was reported, and some aspects of its anaerobic versatility were presented. Earlier reports (Obueckwe et al., 1981; Semple and Westlake, 1987) suggested that a

KENNETH H. NEALSON AND BRENDA LITTLE

220

TABLE 111

PHYLOCENETIC AFFILIATIONS OF METAL REDUCERS Archaea

No known representatives

Bacteria Gram-positives

Gram-negatives gamma'

delta"

epsilon' novelh novel"

Bacillus spp. Bacillus infernus Bacillus spp. SG-1

R u s h et a]., 1994 Boone et al., 1995 DeVrind eta]., 1986

Shewanella putrefaciens Shewanclla alga Ferrirnonas baleurica Aerornonas spp.

Myers and Nealson, 1988a Cnccavo ef a].,1997 Rosello-Mora et al., 1995 Nealson, unpublished

Geobacter rnetallireducens Geobacter acefoxidans Desulfurornonas acetoxidans Pelobncter carbinolicus Desulfurornusa spp.

Lovley eta]., 1993a,b Lonergan et al., 1996 Roden and Lovley, 1993 Lonergan et a/., 1996 Lonergan et a[., 1996

Geospirillum barnesi

Lonergan et al., 19%

Geothrix fermentens

Coatesh

Geovibrio ferrireducens

Caccavo et a]., 1997

'Among the domain bacteria, three groups of the proteobacteria have members that are metal reducers. "IJnpublished results cited in Lonergan et ol., 1896.

strain of this same species was a dissimilatory iron reducer, but no demonstration of growth at the expense of metal reduction was made. Lovley and Phillips (1988) reported the isolation of Geobacter metallireducens (then referred to as GS-15), an obligate anaerobic iron reducer that grew with Fe3+ as the sole electron acceptor and actively reduced Mn4+. For several years, these two organisms were the only two dissimilatory metal reducers studied, but the situation has changed markedly in the past few years, with the isolation of many new strains and species. A summary of known metal reducers (Table 111) shows their diversity and their phylogenetic affiliations. This field is in its infancy, and one can expect that this already broad group will grow substantially.

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

221

The largest group of metal reducers known to date are obligate anaerobes that can be placed in the Geobacter group, as defined by Lonergan and colleagues (1996). This group includes a number of bacteria, most of which share the ability to reduce elemental sulfur to sulfide. The group is in the delta group of the Proteobacteria, and is aligned with sulfate-reducing bacteria (SRB) such as Desulfovibrio spp., some of which have also been reported to be iron reducers (Coleman et al., 1993), although not proven to grow at the expense of iron reduction. The major work on biochemistry and physiology has been limited to G. metallireducens (Lovley et al., 1993a). G. metallireducens grows with lactate, acetate, and a variety of other carbon sources, requires cell contact with solid iron oxides to effect reduction, and can reduce a number of other inorganic electron acceptors, including nitrate and U6+ (Lovley et al., 1991). S. putrefaciens, and a closely related species Shewanella alga (Caccavo et al., 1992), are facultative organisms that grow not only on oxygen and metals as electron acceptors, but display a remarkable versatility-some strains are able to reduce and grow on 10 or more different electron acceptors. Many MRB strains can also reduce elemental sulfur (polysulfide), an unusual ability for an aerobe (Perry et al., 1993; Moser and Nealson, 1996), and produce sulfide from the anaerobic reduction of thiosulfate. These organisms require surface contact for the reduction of solid metal oxides (Arnold et al., 1988), colonize the surfaces during reduction (Figs. 2 and 3; Little et al., 1997a), and have markedly different rates of metal dissolution on different metal oxides (Burdige et a]., 1992; Little et al., 1997a). Unlike G. metallireducens, S. putrefaciens is unable to grow on acetate anaerobically, but it does grow with formate as the sole source of carbon and energy (Scott and Nealson, 1994), and utilizes H2 as an energy source for metal reduction (Lovley et a]., 1989a). IV. Reduction of Metals by Iron and Manganese Reducers

A. GENERALFEATURES

Although the study of dissimilatory microbial metal reduction is a new field, some general features are emerging. Several metals reportedly reduced by MRB are shown in Table IV, and some of these may be environmentally significant for both biodegradation and bioremediation (Fredrickson and Gorby, 1996). Whether catalysis reactions are specific and/or independent is, in general, not known, although for S. putrefaciens mutants have been obtained that are deficient in

222

KENNETH H. NEALSON AND BRENDA LITTLE

F I G . 2. Imagns of cells of Shewonella putrefaciens growing on manganite (MnOOH). Top panel (a) shows uninoculated MnOOH imaged by environmental scanning electron microscopy (ESEM].Middle pariel (b) shows ESEM image of MnOOH (after 90 h of growth with S. putrefaaens MR-4) with bacteria that are obscured by the presence of extracellular polymer. Bottom panel (c) shows the same field as in the middle panel, but after sample drying (polymer dehydration) and imaging by standard scanning electron microscopy (SEM).

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

22 3

224

KENNETH H. NEALSON AND BRENDA LITTLE TABLE IV METALS REDIJCED BY IRON/MAN~;ANESE-~DUCINC BACTERIA

Metal Fe3+

Form or mineral name Soluble chelate (EDTA, NTA)" Fe(OH)? (ferrihydrite)

Reference

FeaO4 (magnetite) Fe-rich clays (smectite)

Arnold et al. (1986) Arnold et 01. (1986) Roden and Zachara (1996) Little e t a ] . (1997a,b) Roden and Zachara (1996) Little eta]. (1997a,b) Roden and Zachara (1996) Kostka and Nealson (1995) Kostka et al. (1996)

Mn3+

Soluble chelate (pyrophosphate) MnOOH (manganitc)

Kostka et al. (1995) Larsen et al. (in review)

Mn4'

MnOz (amorphous) MnOz (birnessite) MnOz (6-MnOz) MnOz (pyrolusite)

Burdige et al. (1992) Burdige et al. (1992) Burdige et 01. (1992) Burdige st a]. (1992)

u6+

Soluble UO$

Lovley eta!. (1991)

Ih+

Soluble 10;'

Ferrenkopf e i al. (in press)

Crb+

Solrible CrO;'

Lovley el al. (1993b)

co3+

co '+-EDTA

Lovley eta]. (1993b)

FeOOH (goethite) Fez03 (hematite)

"EI)TA = ethylenntliarnine tctramxtic acid: N'I'A = nitrilo triacetic acid

Fe but not Mn reduction, and vice versa, thus implying that the terminal reductases for the two processes are different (see Nealson and Saffarini, 1994). Similar mutants have not been reported for other metals, or for other MRB. Growth coupled to reduction of metals has been demonstrated only for Fe and Mn, with one report of growth of G. metallireducens on UG+ (Lovley et al., 1993b). In general, the abundance of the other metals is very low, on the order of ppm or less, so that it is not easy to imagine the evolution of specific systems to harvest such minuscule amounts of energy. While dissimilatory reduction of selenium is known to be a bacterial process (Oremland et a]., 1994), to our knowledge, none of the h4RB have been shown to reduce selenium.

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

225

Burdige et al. (1992) showed that a culture of S . putrefaciens growing on a series of manganese oxides with different mineralogies gave very different rates of reduction. In general, the rates of reduction were proportional to metal oxide surface area, so that a highly crystalline oxide like pyrolusite (with a very low surface-to-volume ratio) was reduced very poorly: almost not at all. Roden and Zachara (1996) studied the relationship between iron oxide reduction and surface area, concluding that the rate and extent of iron oxide reduction were controlled by the surface area and site concentration of the solid phase. From studies such as these, it seems likely that the major controlling factor in solid metal reduction is surface area, with such other factors as crystal structure, morphology, free energy, and particle aggregation participating to lesser degrees (Roden and Zachara, 1996). Yield studies of bacteria on solid substrates are difficult because of the attachment of the bacteria to the oxides (Figs. 2 and 3). When growing on manganese oxides (Fig. 21, S. putrefaciens forms a layer of extracellular polymer that obscures individual cells when viewed by environmental scanning electron microscopy (ESEM). The nature of this polymer is not yet elucidated, but it is likely to be a polysaccharide based on the fact that it is difficult to visualize when samples are dehydrated and viewed by standard scanning electron microscopy (SEM) (Fig. 2). Interestingly, when the same cells are grown on iron oxides, no extracellular polymers are conspicuous (Fig. 3) when examined by ESEM.

B. BIOCHEMISTRYOF METAL REDUCTION As of this writing, iron or manganese reductases have not been purified or characterized, although high levels of both activities have been observed in whole cells as well as in cell-free extracts. Myers and Myers (1992, 1993) reported that cytochromes and the iron reductase activity of S . putrefaciens are located in the outer membrane, consistent with the observations that metal oxides are solids and that cell contact is required for metal reduction. Tsapin et al. (1996) purified a small (12 kDa) tetraheme cytochrome cg of very low potential (-233 mV) with a high sequence similarity to the cytochrome cg of Desulfovibrio desulfuricans, and showed that this cytochrome in its reduced state could reduce Fe3+. Pleiotropic mutants missing this cytochrome are unable to reduce iron and several other electron acceptors (Lies and Nealson, unpublished). Despite this circumstantial evidence, the proof that this cytochrome is actually the iron reductase has not been presented. However, the gene is now cloned and sequenced

226

KENNETH H. NEALSON AND BRENDA LITTLE

1 ('**

(SRB)

$2032-

(CH20)n

5042-

Frc:. 4. Direct and indirect reduction of Mn4+.'This diagram shows the ways in which S . putrefaciens can reduce manganese. The solid lines indicate reactions that are catalyzed primarily by bacteria, while the squiggly lines indicate those reactions that occur by inorganic chemical transformations. MRB = metal-reducing bacteria. SRB = sulfur(sntfate, thiosulfate, or elemental sulfur) reducing bacteria.

(Tsapin et al., 1997), so it should be possible to generate insertional mutants and specify a function for this cytochrome. In addition, five other c-type cytochromes have been purified from S. putrefaciens, two of which have low redox potentials consistent with metal reductases, and one of which is membrane-bound (Tsapin et a].,1997). Lovley et ul. (1993b) partially purified a cytochrome c3 from Desulfovibrio vulgaris that could reduce U", and concluded that it was the U reductase. In many cases of metal reduction, the possibility that indirect reactions occur must be considered. Manganese is a particular problem because it is so easily reduced by other reductants (Stone, 1987a,b) as outlined in Fig. 4 (Myers and Nealson, 1988b). Fez+and HS- are both produced by S. putrefaciens and can reduce Mn4+.Thus, any sulfur- or iron-reducing bacterium can appear to be an Mn reducer via indirect reactions, and, in fact, with catalytic amounts of iron or So, a biogeochemical cycle resulting in Mn reduction can be established. For these reasons, the identification of microbes as Mn4+ reducers should be regarded with suspicion until mutants are produced that show the separation of direct and indirect reduction.

c.

REGULATION OF

METAT. REIXJCTJON

Studies by King (1990) showed that addition of Fe3+or Mn4+resulted in inhibition of sulfate reduction or methanogenesis in sediments, suggesting that these substrates were preferred electron acceptors for sediment communities. Earlier work by Burdige and Nealson (1986)

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

227

demonstrated that pure cultures of sulfate reducers continued to produce sulfide in the presence of Mn4+,reducing the Mn4+via sulfide production. Thus, the relationship between metal respiration and respiration of sulfate or sulfur is probably not simply one of the physiological response of single organisms, but may represent a complex community response, perhaps even a competition for substrates. In fact, if sulfate reduction is assessed by measurement of sulfide, then the effect of adding Mn4+could be totally directed at the product of the reaction rather than the process itself, that is, sulfate reduction would be apparently inhibited (because of the lack of sulfide production), while the process could be proceeding at the same rate (see Fig. 4). To our knowledge, work with pure cultures to address such questions has not been initiated. Several studies of regulation of metal reduction by S. putrefaciens indicate that regulation occurs at several levels. Arnold et al. (1990) and DiChristina (1992) concluded that a physiological competition exists between iron and other electron acceptors, with oxygen or nitrate capable of inhibiting iron reduction. Similarly, Myers and Nealson (1988b) showed that oxygen or nitrate inhibited reduction of Mn4+by S. putrefaciens, but that less electropositive electron acceptors like fumarate or sulfite had no such inhibitory effect. It appears that metal reduction follows thermodynamic rules for a respiratory substrate. In the presence of oxygen or nitrate, no metals are “breathed,” but in the absence of other good electron acceptors bacteria use iron or manganese for respiration. Interestingly, some substrates like iron (which is converted to Fez+)or sulfur (which is converted to HzS) result in apparent increases in Mn4+respiration via indirect reactions, discussed above (Fig. 4). Anaerobic respiration in bacteria is controlled by a variety of regulatory systems (fnr, arcBA, etc.) that sense oxygen, or some product of oxygen, and control metabolic pathways (like the tricarboxylic acid cycle) and specific reductases at the level of biosynthesis (Spiro and Guest, 1990; Lin and Iuchi, 1991; Unden et al., 1995). Mutants in these control systems characteristically result in pleiotropic phenotypes, with a number of different anaerobic processes being simultaneously affected. Several such pleiotropic mutants have been isolated from strains of S. putrefaciens (DiChristina and DeLong, 1994; Saffarini and Nealson, 1993; Saffarini et a]., 1994), and in one case the gene controlling the mutation was isolated, sequenced, and found to be an analogue of the fnr gene from E. coli (Saffarini and Nealson, 1993), suggesting that similar control mechanisms operate to regulate the anaerobic respiration of this metal reducer. Similar studies of regulation at the molecular level have not been reported for other metal reducers.

228

KENNETH H. NEALSON AND BRENDA LITTLE

pyrite FeS, (greigite FeC03 (siderite)

Fe (OH)3 (ferrihydrite) Fe,Os (hematite) FeOOH (goethite)

Mn'2 birnessite manganates MnOOH(manganite)

Mn02

(CH20)n Mn4+ (Mn3+)

CO2

MnC03 (rhodochrosite)

4 Uk Mn'+ (MRB)

Soluble Mn"

FIG.5. Fates of soluble Mn and Fe produced under anoxic conditions. Manganese docs not easily form insoluble sulfides or mixed phase oxides. If COZ production is high, rhodochrosite may form: otherwise, MnZ+ remains as the soluble salt and difhises upwards to the oxygen interface. Depending on the chemistry of the anoxic environment, Fez+may have any of a variety of "fates."

D. PRODUCTS OF METALREDUCTION For the most part, the products of metal reduction are soluble forms of the metals, leading to an increase of Fez+or Mn2+in the anoxic zones, but in laboratory studies, a variety of other end-products are seen, depending on experimental conditions (Fig. 5). MnZt tends to be fairly unreactive with regard to end-products, and unless experiments are conducted in closed tubes with high levels of organics (which raises the level of carbon dioxide in the water), the product is primarily dissolved manganous ion. In closed tubes, a pinkish-white mineral phase called rhodochrosite (MnCO,) is formed. In many rhodochrosite samples in nature, the pink rhodochrosite strata are interlayered with white carbonates, suggesting episodic introduction of MnZt. The solubility of manganese sulfide is such that this mineral does not form in laboratory experiments, and is rarely found in nature. Ferrous (Fez+)iron is a much more reactive sedimentary component, rapidly forming a variety of insoluble sulfides, including pyrite. In marine systems, insoluble iron sulfide formation leads to scavenging of the iron from the system, while in freshwater systems with very low sulfate concentrations iron often remains in the system as a major redox

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

229

species. In addition to iron sulfides, siderite or iron carbonate (FeC03) can form, especially in closed-tube laboratory experiments where COz increases as a result of respiration. As shown in Fig. 5, reduced iron can interact with Mn4+ to reduce it, thus being reoxidized to Fe3+.This reaction in part explains why the zone of iron reduction is always found below the zone of manganese reduction. Magnetite is one of the more poorly understood mineral phases in terms of the role of dissimilatory iron reducers. Several authors have noted that magnetite is an extracellular product of iron reducers (Bell et a]., 1987; Lovley et a]., 1987; Roden and Lovley, 19931, routinely formed in experiments where chelated iron (as iron citrate) is used as the source of Fe3+.However, if the pH is kept at 7.0 or below and the concentration of magnetite is kept low, it is possible, in the presence of organic matter, for S. putrefaciens to catalyze the further reduction of magnetite to Fez+(Kostka and Nealson, 1995). It is not known whether such reactions are important in the sedimentary budget of magnetite: its formation, dissolution, or both. V. Electron Transport In Metal Reducers A. CYTOCHROMES

Only a limited amount of work has been reported with components of the electron transport systems of the anaerobic metal reducers. Studies of cell-free extracts have shown that G. metallireducens and Desulfuromonas acetoxidans contain c-type cytochromes that can be reoxidized by a number of different substrates. The cytochromes c from G. metallireducens could be oxidized by Fe3+,U6+,NO;, Hg, Au, Ag, or Cr6+(Lovley et al., 1993b), while those of D. acetoxidans were oxidized by So, malate, Fe3+,or Mn4+(Roden and Lovley, 1993). Some strains of D. desulfuricans are known to reduce Fe3+and U6+ (Coleman et al., 1993),although they do not couple this reduction to cell growth. Desulfovibrio vulgaris cells can reduce uranium, apparently via a cytochrome c3,which can catalyze the reduction of Fe3+,UG+, or Cr6+(Lovley et al., 1993b3).Naik et al. (1993)isolated three c-type cytochromes (28,46, and 68 kDa) in the partially purified preparation of nitrate reductase from G. metallireducens. In S. putrefaciens, spectral analysis of whole cells and extracts (Obueckwe and Westlake, 1982) showed that c-type cytochromes were present, and that the concentration increased with addition of iron to the growth medium. Arnold et al. (1986) also reported the presence of c-type cytochromes, and noted low oxygen tension-stimulated cyto-

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chrome synthesis. Myers and Myers (1992) concluded that many c-type cytoclrromes were present and that they were primarily localized in the outer membrane fractions. Morris et al. (1990) resolved nine different c-type cytochrome bands by DEAE-Sepharose chromatography. In the above studies, cytochromes were not purified, nor were they characterized in detail. In more recent definitive work, a large (63.8 kDa) soluble tetraheme flavocytochrome c was purified and identified as a fumarate reductase (Morris et al., 1994; Pealing et al., 1995). Heme c replaces iron sulfur centers characteristic of the membrane-bound funiarate reductases of other organisms. The heme midpoint redox potentials are -220 and -320 mV. The gene coding for this protein has been isolated and sequenced (Pealing et al., 1992). Tsapin et al. (1996) recently purified and sequenced (see Tsapin et al., 1997) a small (12.8 kDa) c3-type cytochrome from S . putrefaciens. This molecule, like the flavocytochrome of Morris et al. (1994), has a very low redox potential of -233 mV and is a tetraheme molecule with structural similarities to the cytochromes c3 from D. acetoxidans and the cytochrome portion of the flavocytochrome reported by Morris et al. (1994). The gene for this protein has been cloned and sequenced, revealing a leader sequence for transport to the periplasm (Tsapin and Nealson, unpublished data).

B. QUINONES No detailed quinone analyses have been presented from G. metallireducens, but on the basis of difference spectra of lipophilic extracts it was concluded that the quinones are menaquinones and that the types and levels are similar to those found in sulfate and sulfur reducers (Lovley et a]., 1993a). Not surprisingly, the facultative S. putrefaciens has a wide variety of quinone types, and these vary extensively between aerobically and anaerobically grown cells. The aerobic cells contain two ubiquinones, (1-7 and Q-8, and one menaquinone, MK-7, along with a small level of a methylated menaquinone, MMK-7. Under anaerobic conditions, irrespective of electron acceptor, the major quinone was MMK-7, with Q-7, Q-8, and MK-7 being minor types. Several other quinones appeared under anaerobic conditions, including several MMK types (Lies et al., 1996; Sano eta]., 1997). Under microaerophilic conditions, some strains produced 15 or more different quinones.

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VI. Metal-Reducing Bacteria in Natural Environments

MRB have been isolated from a variety of environments, including marine and lake sediments, and oil-field injection waters (Nealson and Saffarini, 1994; Lonergan et al., 1996). In addition to their role in the biogeochemical cycling of carbon, the ability of MRB to reduce a variety of different substrates can lead to environmental consequences and problems, including corrosion, release of bound metals or radionuclides, and a change in the physical properties of clays. Conversely, reactions related to metal reduction have potential for exploitation in areas of bioremediation. A. CORROSION

While it has been established that the most devastating microbiologically influenced corrosion (MIC) takes place in the presence of microbial consortia in which many physiological types of bacteria interact in complex ways, dissimilatory iron and/or manganese reducers represent one of the important components of the complex corrosion community (Fig. 6). Obueckwe et al. (1981a,b) used polarization studies to demonstrate that a Pseudomonas (Shewanella)isolate from an oil field caused anodic polarization of mild steel coupons accompanied by conversion of ferric to ferrous compounds. There was a loss of passivity and an intense depolarization of the anode in the presence of the organism. Electron micrographs demonstrated that a dense crystalline surface deposit covering the uninoculated metal was removed when the organism was introduced. Furthermore, the presence of the organism prevented formation of a protective ferric layer. Obueckwe et al. (1987) recognized that Shewanella influenced corrosion by the reduction of Fe3+to Fez+and S20,2-to S2-. In experiments designed to inhibit one or both of these reduction reactions, both the production of S2- and Fez+ simultaneously and the production of Fez+ alone by bacteria were responsible for anodic dissolution of the carbon steel. When only S2was produced, the initial increase in anodic reaction was due to reactions of the S2- with the metal. The resultant FeS eventually protected the metal, followed by a decrease in the anodic reaction. Corrosivity of soils has been associated with soluble iron, and soils with an Fez+ content above 333 pg-* are very corrosive (Booth et al., 1967). Little et al. (1997a) used electrochemical noise to demonstrate the aggressive attack of carbon steel by pure cultures of S. putrefaciens and to further differentiate mechanisms of iron and thiosulfate reduction.

232

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I

N

s"

I &

g

KENNETH H. NEALSON AND BRENDA LITTLE

f

-

.-d

a

BREATHING MANGANESE AND IRON: SOLID-STATE RESPIRATION

23 3

Little et al. (1997b) used synthetic iron oxides (goethite, FeOOH; hematite, Fe,O,; and ferrihydrite, Fe(OH),) as model compounds to simulate passivating films on carbon steel to demonstrate that corrosion of carbon steel by S. putrefaciens was related to surface mineralogy. Dissolution of these oxides exposed to pure cultures of the metal-reducing bacterium was followed by direct measurement of ferrous iron, using atomic absorption spectroscopy (AAS), coupled with microscopic analyses. Bacteria colonized all mineral surfaces and formed biofilms within 48 h. After 190 h, confocal laser scanning microscopy (CLSM) images (Fig. 3) have been used to show that bacteria penetrate both goethite and ferrihydrite but are restricted to the surface of hematite even after 200-h exposure (Little et al., 1997a).

B. RELEASE OF ADSORBED POLLUTANTS Iron and manganese oxides are regarded as the “scavengers of the sea” (Goldberg, 1954)because of their ability to adsorb other metals and trace components. Interaction of metal oxides with trace metals is considered to be of major importance in sediment and water column chemistry (Balistrieri and Murray, 1982, 1984; Tessier et al., 1996). Manganese oxide fibers have been used to collect radium from seawater (Moore, 1975), and metal oxides have been proposed as a means for disposal of radionuclide waste (Mott et al., 1993).As a consequence, when iron or manganese oxide reduction occurs in municipal water systems, not only is the water fouled by excess soluble manganese and/or iron, but trace components bound to the metal oxides may also be released (Francis and Dodge, 1990). Such reactions may account for the distribution of trace metals (Francis and Dodge, 1990; Rose et al., 1993; Tessier et ~ l . , 1996) and radionuclides like uranium in sediments (McKee eta]., 1987) and anoxic water columns (McKee and Todd, 1993). C. CLAYREDUCTION

Kostka et QI. (1996) reported reduction of structural iron from smectite clays by strains of S . putrefaciens. Reduction of the clay is accompanied by changes in color, swellability, and other properties (J. Stucki, personal communication), suggesting a major impact in anaerobic soils or sediments where it occurs. The environmental importance of such reactions is not known, but the knowledge that bacteria can couple organic carbon oxidation to reduction of clays should be considered in studies of anaerobic sediments.

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D. BIOREMEUIATION Metal-reducing bacteria may offer potential applications in bioremediation, including degradation of toxic organic pollutants. Because of the reasonably high redox potentials of Feat and Mn4+,organisms that use them are relatively efficient at converting organic carbon pollutants into harmless COz and organic byproducts that can be metabolized by other anaerobic bacteria. G. rnetaffireducenscan oxidize certain aromatics under anaerobic conditions using iron as the oxidant (Lovley et nf., 1989b, 1990; Kazumi et al., 1995). The direct approach to bioremediation has the following advantages over using more standard respiring bacteria (e.g., aerobes, nitrate reducers, or sulfate reducers): (1) iron oxides are solids and can be delivered to a contaminated site without the possibility of their diffusing away; (2) iron oxides are rather specific substrates, as far as is known, so that competition from other bacteria for the electron acceptor should be minimal; and (3) in stratified aqueous environments reduced iron should diffuse upwards, be reoxidized by molecular oxygen in the overlying oxic zone, and returned to the anoxic zone via gravity, thus acting as a “pump” for oxidizing equivalents, as proposed by Nealson and Myers (1992). The introduction of MRB and their potential in pollutant removal for both the short and long term might be very high, especially if iron reducers not inhibited by oxygen were available. Two reports suggest that Shewnneffa spp. can donate electrons to chlorinated hydrocarbons, thus reductively dechlorinating toxic compounds by converting tetrachloromethane to trichloromethane (Picardel et al., 1993; Petrovskis et a]., 1994). No other chlorine transformations were observed, and the reaction was inhibited by oxygen, but not by other electron acceptors. These results are consistent with a fortuitous reduction by reduced c-type cytochromes under anaerobic conditions (Picardel et af., 1993). Ferrous iron, especially in the presence of surfaces, can catalyze the reduction of nitro groups on substituted nitrobenzenes (Klausen et al., 1995) and other nitroaromatic compounds (Heijman et nl., 1995). Thus, any organism that can produce Fez+can potentially catalyze the reduction of toxic nitrates. In reduction of the pollutant, iron is reoxidized, so that a catalytic amount of iron could be used to cycle between the oxidized pollutants and respiring bacteria (Fig. 7). Metals can be removed from solution by indirect reactions via the production of sulfide and resulting production of insoluble metal sulfides. Many metal-reducing bacteria are also capable of generating hydrogen sulfide through the reduction of sulfate (Desuffovibrio),sulfur

xFe2

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6

2 35

“bound

“bound Fe3+

FIG.7. Degradation of nitroaromatics by Fe-reducing bacteria. This figure, modeled after that presented by Heijman et al. (1995), shows the inorganic reduction of nitroaromatics. The process is strongly enhanced if surfaces such as minerals or clays are present, indicated by the “bound” iron.

(Desulfuromonas, Shewanella), or thiosulfate (Shewanella). Toxic and trace metals can be removed via precipitation as insoluble sulfides, which have very low solubility products (Stumm and Morgan, 1981). Fude et al. (1994) demonstrated Cr6+reduction/detoxification via H2S by a consortium of SRB. Metals can also be removed via direct reduction by the MRB. While iron and manganese are solubilized, other metals are converted to insoluble forms upon reduction. Of note are chromium (Cr6+)and uranium (U6+),which are soluble in oxidized form, but insoluble as the respective Cr3+and U4+ reduced species. Reduction of U6+ has been demonstrated for both G. metallireducefis and S. putrefaciens (Lovley et al., 1991), and has been proposed as a mechanism for concentrating and thus removing radionuclide waste. The cytochrome c3 from G. metallireducens can reduce U6+ (Lovley et d.,1993b), presenting the possibility of immobilized enzyme treatment of waste materials. In this regard, the recent cloning of cytochrome cgfrom S. putrefaciens (Tsapin et al., 1997) could offer a ready supply of protein for such work. Chromium reduction has also been reported for D. vulgaris, and the cytochrome c3 partially purified from this organism is capable of Cr6+ reduction. S. putrefaciens can reduce Cr6+,but no detailed studies have been presented (Nealson, unpublished). As with uranium, the removal of toxic chromium should be possible using either intact cells or cellfree systems of MRB. VII. Summary

Respiration of solid compounds was, until a few years ago, thought to be very unlikely, and, although metal reduction was known to occur, organisms that coupled growth and respiration to reduction of oxidized

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iron or manganese were not known. In recent times, this view has changed, and several groups of microbes have been described that live via dissimilatory reduction of metal oxides. MRB represent a fairly wide range of phylogenetic types, although to date no archaea are included among them. They often share the property of being able to reduce elemental sulfur, a trait that was previously unknown in organisms able to grow aerobically. The ability to “breathe” metal oxides adds a new, not previously considered dimension to bacterial respiration. How can an organism transfer electrons to solid substrates? While this question is not yet answered, many recent advances have been made in both the biochemistry and molecular biology of some metal reducers. Of particular note are reports of reductases and cytochromes localized to the outer membranes of the MRB, and isolation of c-type cytochromes with low potentials. Since iron and manganese oxides are solids, their reduction has environmental consequences of both decreasing particulate load and increasing levels of dissolved iron and manganese. In addition, if other components adsorb to the metal oxides, as often occurs in nature, they may be released by the metal reduction. Microscopic studies of metal oxides during microbial reduction show contact of the organisms with surfaces, and distinct environmental responses between organisms grown on manganese versus iron oxides. Finally, the unique abilities of these organisms present both environmental problems and opportunities. Problems resulting from metal oxide reduction include corrosion, alteration of sediment and soil properties, release of toxic adsorbed compounds in sediments, and fouling of public water supplies with MnZ+and Fez+during periods of anoxia. Opportunities involve new approaches to bioremediation: anaerobic consumption of organic pollutants, removal of toxic metals either by sulfide precipitation or by conversion to insoluble reduced forms, and addition of Fez+to environments where it may be active in reduction of nitroaromatics. ACKNOWLEDCMENTS

KHN wishes to thank NASA (exobiology), the NSF (chemical oceanography), and the Wisconsin Sea Grant Program for support for his research on this topic. He also gratefully acknowledges the Office of Naval Research for the Distinguished Visiting Researcher Award during 1996, contract no, NO00149 96-J-06. BJL acknowledges support by the Office of Naval Research, contract no, NO001497 WX30031. We thank the following NRL personnel: Richard Ray for ESEM and CLSM micrographs; and Darlene Jorns, Maria Banker, and Mary Ellen Turncotte for help with the figures. This document is NRL Contribution Number NRLIBAI 73 33-97-0004.

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